Nestin knockout leads to embryonic lethality and self-renewal deficiency in neural stem cells (NSCs). However, how nestin maintains self-renewalremains uncertain. Here, we used the dosage effect of nestin in heterozygous mice (Nes+/-) to study self-renewal of NSCs. With existing extracellularsignaling in vivo or in vitro, nestin levels do not affect proliferation ability or apoptosis when compared between Nes+/- and Nes+/+ NSCs. However,self-renewal ability of Nes+/- NSCs is impaired when plated at a low cell density and completely lost at a clonal density. This deficiency in self-renewal at a clonal density is rescued using a medium conditioned by Nes+/+ NSCs. In addition, the Akt signaling pathway is altered at low density and reversed by conditioned medium. Our data show that secreted factors contribute toward maintaining self-renewal of NSCs by nestin, potentially through Akt signaling.